Newly replicated DNA molecules of herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) consist of head-to-tail concatemers arising most likely by rolling circle replication. The large concatemers are subsequently cleaved to generate unit length molecules which are packaged into viral nucleocapsids. Cleavage occurs within the a sequences situated at the junctions between adjacent viral genomes in the concatemers. In previous studies under this project we have employed constructed defective virus genomes to investigate the mechanisms of concatemeric cleavage. Constructed defective genome concatemers were generated by replication of seed monomeric repeats containing a viral DNA replication origin and an a sequence, containing a cleavage/packaging signal. These studies have revealed that concatemeric cleavage was coupled to the packaging of viral DNA. Analyses of the structural features of concatemeric junctions and generated termini in packaged and free nuclear and cytoplasmic DNAs have yielded evidence concerning details of the cleavage/packaging process, including the recognition of two signals located within the a sequence which play distinct roles in the cleavage/packaging; the amplification of a sequences which is coupled to cleavage, the scanning of the viral DNA concatemers during packaging for a directly repeated a sequence containing the terminal cleavage signal, and the requirement for full length encapsidated DNA genomes for further maturation of nuclear capsids into enveloped cytoplasmic virions. On the basis of the data two models were formulated to explain the known properties of a sequences in standard as well as defective virus DNA. We now propose three sets of studies to continue our analyses of early events in the packaging process. In the first set of studies we will continue our investigations of the mechanism of concatemeric DNA cleavage, through testing and refining various features of the models proposed. In the second set of studies we will continue our analyses of in vivo intermediates in the packaging process. The third set of studies will attempt to develop an in vitro system for the cleavage and possibly encapsidation of HSV DNA. It is expected that these studies will uniquely increase our understanding of events constituting the culmination of the infectious cycle, and involving highly specific DNA-protein and protein-protein interactions.